Method of extracting and method of purifying an effective substance

ABSTRACT

A method for extracting a hydrophobic group-containing water-soluble organic compound, comprising the step of bringing an aqueous solution containing the hydrophobic group-containing water-soluble organic compound and a saccharide into contact with a polar organic solvent to obtain an aqueous phase and an organic phase, whereby the hydrophobic group-containing water-soluble organic compound is transferred to the organic phase. The saccharide concentration of the aqueous solution may be at least 12 g per 100 ml of the aqueous solution. The aqueous solution may further contain a phase separation assisting agent. The phase separation assisting agent may be selected from the group consisting of sodium chloride, sodium citrate, magnesium sulfate, and ammonium sulfate.

TECHNICAL FIELD

The present invention relates to a method for extracting a hydrophobicgroup-containing water-soluble organic compound from an aqueous solutioncontaining the hydrophobic group-containing: water-soluble organiccompound (e.g., an extract derived from an animal or a plant as well asan enzyme reaction solution) with high purity and high yield.

BACKGROUND ART

As represented by crude drug extracts, a number of natural compoundshaving a variety of physiological activities are known. These naturalcompounds are also called physiologically active substances. A number ofphysiologically active substances are generally purified by conductingextraction using a material containing the physiologically activesubstance and water, warm water or low-concentration aqueous alcoholsolution to obtain an extract solution, concentrating the extractsolution, and subjecting the concentrated extract solution to columnchromatography. However, such a purifying method requires a large columnand equipment accompanying therewith in order to produce a large amountof a physiologically active substance. A small column has very poorefficiency. Therefore, a purified physiologically active substance isvery expensive.

Attempts have been made to purify physiologically active substances by asolvent extraction method. However, a method of adding an organicsolvent which is inherently immiscible with water, such as ethylacetate, butanol, and chloroform, to an aqueous solution, stirring,allowing the solution to stand to obtain two phases, i.e., aqueous phaseand organic solvent phase, and recovering the physiologically activesubstance transferred to the organic solvent phase, cannot be used forfoods due to safety problems. Even when a physiologically activesubstance is used for applications other than foods, somephysiologically active substances are inefficiently extracted using anorganic solvent since the physiologically active substances are notsignificantly transferred to the organic solvent phase. Since someorganic solvents which can be used for foods, such as ethanol andacetone, are miscible with water, these organic solvents cannot be usedto extract and purify a physiologically active substance from aqueoussolution.

Hesperidin is a representative flavonoid which is contained in orangejuice. Flavonoids represented by hesperidin are known to havephysiological actions described below, for example. Hesperidin and rutinwere previously called vitamin P, and have long been known to have anaction that lowers blood pressure (Shintaro Kamiya, Shin-Vitamin-Gaku,[New Vitamin Study] (The Vitamin Society of Japan) 1969, p 439). It hasalso been reported that hesperidin has the following physiologicalactions: anti-inflammatory action, analgesic action (E, M. Galati etal., Il Farmaco, 49, 709-712(1994)), antiallergic action (HideakiMatsuda et al.; Yakugaku Zasshi [Journal of Pharmacology], 111,193-198(1991), J. A. Da Silva Emim et al.; J. Pharm. Pharmacol., 46,118-712(1994)), an action that reduces LDL-cholesterol to ameliorateblood cholesterol levels (M. T. Monforte et al.; Il Farmaco, 50,595-599(1995)), and carcinostatic action (T. Tanaka, et al.; CancerResearch, 54, 4653-4659(1994), T. Tanaka, et al.; Cancer Research, 57,246-252(1997), T. Tanaka, et al.: Carcinogenesis, 18, 761-769(1997), T.Tanaka, et al.: Carcinogenesis, 18, 957-965(1997)). Further, in recentstudies, it is expected that hesperidin also has an action that promotesdifferentiation of fat precursor cells to ameliorate conditions, such asdiabetes. Diosmin has a vigorous antioxidant activity.

A medical agent containing Diosmin and hesperidin is utilized as atherapeutic drug for venous insufficiency, hemorrhoids, and the like (C.Labrid: Angiology, 45, 524-530(1994)). It has also been reported thathesperidin alone, Diosmin alone, and a mixture of hesperidin and Diosminsuppresses oral cancer, esophageal cancer, colorectal cancer, and thelike (T. Tanaka, et al.; Cancer Research, 54, 4653-4659(1994), T.Tanaka, et al.; Cancer Research, 57, 246-252(1997), T. Tanaka, et al.;Carcinogenesis, 18, 761-769(1997), T. Tanaka, et al.; Carcinogenesis,18, 957-965(1997)).

Naringin and neohesperidin are known as bitter substances of citrus, andare used in foods and beverages for the purpose of providing bitterness.

Further, it has been recently revealed that isoflavone effectivelyimproves bone density, suppresses occurrence of breast cancer, and thelike (Toda et al. Foods and Ingredients Journal of Japan, No. 172, 83-89(1997)).

Hesperidin and rutin are inherently insoluble in acetone.

On the other hand, flavonoids, such as hesperidin, naringin,neohesperidin, and rutin, are poorly soluble in water. In order toovercome this drawback, i.e., the poor solubility, attempts have beenmade to efficiently solubilize these poorly soluble compounds. Forexample, a method of improving the solubility of flavonoids, such ashesperidin, naringin, neohesperidin, and rutin, by enzymaticglycosylation, is known (Japanese Laid-Open Publication No. 7-107972).

A method of improving the solubility of catechin, caffeic acid, kojicacid, hydroquinone, catechol, resorcinol, protocatechuic acid, gallicacid, vanillin, daidzein, genistein, α-resorcylic acid andphloroglucinol other than the above-described flavonoids by enzymaticglycosylation for the same purpose, is known (Japanese Publication forOpposition No. 7-36758 and T. Nishimura, J. Ferment. Bioeng., 78 (1994)p 37).

However, since the water solubility of the glycoside itself is improved,the glycoside cannot be efficiently extracted in a solvent immisciblewith water. Also, due to safety problems, column chromatography, such asadsorption chromatography, is required for purification of glycosidesfrom enzyme reaction solutions in which glycosylation is conducted.

In conventional purification methods, in order to obtain partiallypurified flavonoids, catechins, phenols and glycosides thereof fromnatural materials, a method of conducting extraction using the naturalmaterial and alkaline aqueous solution, organic solvent, hot water, orthe like and then purifying the extract solution by columnchromatography, has been employed. However, extraction and purificationusing a safe organic solvent which can be used for foods are requiredfor obtaining a large amount of these substances with high yield andwith low cost. However, since acetone which can be utilized for foods ismiscible with water, it is not possible to extract these substancesusing acetone. Most physiologically active substances which areeffective in the food and drug fields have properties such that they areeasily soluble in a high polarity solvent, such as water, ethanol, andacetone, while they are poorly soluble in a low polarity solvent, andtherefore, cannot be efficiently extracted from a low polarity solvent.

DISCLOSURE OF THE INVENTION

As a result of diligent studies, the present inventors have found thateven when using an organic solvent, which is inherently difficult toseparate from aqueous phase where the organic solvent is mixed withwater or hot water, if a substance having water holding capacity, suchas a saccharides is present in an aqueous solution containing aphysiologically active substance, it is possible to easily separateaqueous phase from organic phase after mixing and stirring the aqueoussolution and the organic solvent. The present inventors also found thatthe physiologically active substance was transferred to the organicphase. Further, the present inventors found that by increasing the ionicstrength of the aqueous solution by adding salt (e.g., sodium chlorideand sodium citrate) or an organic acid thereto, an organic solvent whichis not otherwise separated from aqueous phase or which is otherwisedifficult to separate from aqueous phase even if a saccharide is presentcan be separated from aqueous phase, and a physiologically activesubstance can be efficiently transferred to the organic solvent phase.Based on these findings, the present inventors completed the presentinvention.

The method of the present invention is a method for extracting ahydrophobic group-containing water-soluble organic compound, comprisingthe step of bringing an aqueous solution containing the hydrophobicgroup-containing water-soluble organic compound and a saccharide intocontact with a polar organic solvent to obtain an aqueous phase and anorganic phase, whereby the hydrophobic group-containing water-solubleorganic compound is transferred to the organic phase.

In one embodiment, the saccharide concentration of the aqueous solutionmay be at least 12 g per 100 ml of the aqueous solution.

In one embodiment, the hydrophobic group-containing water-solubleorganic compound may be a water-soluble aromatic compound.

In one embodiment, the hydrophobic group-containing water-solubleorganic compound may be selected from the group consisting of phenolderivatives and glycosides thereof.

In one embodiment, the hydrophobic group-containing water-solubleorganic compound may be selected from the group consisting ofhydroquinone glycoside, catechin, salicin, hesperidin, hesperidinglycosides, caffeic acid, salicyl alcohol, and elladitannin.

In one embodiment, the aqueous solution may further contain a phaseseparation assisting agent.

In one embodiment, the phase separation assisting agent may be a salt oran organic acid.

In one embodiment, the phase separation assisting agent may be selectedfrom the group consisting of sodium chloride, sodium citrate, magnesiumsulfate, and ammonium sulfate.

In one embodiment, the polar organic solvent may be tetrahydrofuran oracetonitrile.

In one embodiment, the polar organic solvent may be tetrahydrofuran,acetonitrile, acetone, or isopropyl alcohol.

In one embodiment, the hydrophobic group-containing water-solubleorganic compound may be derived from an enzyme reaction solution.

In one embodiment, the enzyme reaction solution may be a glycosylationreaction solution.

In one embodiment, the glycosylation reaction solution may be ahesperidin or hydroquinone glycosylation reaction solution.

In one embodiment, the hydrophobic group-containing water-solubleorganic compound may be derived from an organism selected from animalsor plants.

In one embodiment the hydrophobic group-containing water-soluble organiccompound may be derived from fruit juice.

In one embodiment, the aqueous solution may be prepared by concentratingan enzyme reaction solution containing the hydrophobic group-containingwater-soluble organic compound and the saccharide.

In one embodiment, the enzyme reaction solution may be a glycosylationreaction solution.

In one embodiment, the glycosylation reaction solution may be ahesperidin or hydroquinone glycosylation reaction solution.

In one embodiment, the aqueous solution may be prepared by concentratingor diluting an extract of an organism, wherein the extract contains thehydrophobic group-containing water-soluble organic compound and thesaccharide, and the organism is an animal or a plant.

In one embodiment, the aqueous solution may be prepared by concentratingfruit juice.

In one embodiment, the aqueous solution may be prepared by adding thephase separation assisting agent to an enzyme reaction solutioncontaining the hydrophobic group-containing water-soluble organiccompound and the saccharide, or a concentrate thereof.

In one embodiment, the enzyme reaction solution may be a glycosylationreaction solution.

In one embodiment, the glycosylation reaction solution may be ahesperidin or hydroquinone glycosylation reaction solution.

In one embodiment, the aqueous solution may be prepared by adding thephase separation assisting agent to an extract of an organism, aconcentrate thereof, or a diluent thereof, wherein the extract containsthe hydrophobic group-containing water-soluble organic compound and thesaccharide, and the organism is an animal or a plant.

In one embodiment, the aqueous solution may be prepared by adding thephase separation assisting agent to fruit juice or a concentratethereof.

The purifying method of the present invention is a method for purifyinga phenol derivative glycoside, comprising the steps of bringing a firstaqueous solution containing a phenol derivative, a phenol derivativeglycoside and a saccharide into contact with a polar organic solvent toobtain a first aqueous phase and an organic phase containing a smallamount of water, whereby the phenol derivative and the phenol derivativeglycoside are transferred to the organic phase, recovering the organicphase containing the small amount of water, removing the polar organicsolvent from the organic phase containing the small amount of water toobtain a second aqueous solution containing the phenol derivative andthe phenol derivative glycoside, bringing the second aqueous solutioninto contact with ethyl acetate to obtain a second aqueous phase and anethyl acetate phase, whereby the phenol derivative is transferred to theethyl acetate phase, recovering the second aqueous phase, andconcentrating and cooling the second aqueous phase to precipitate thephenol derivative glycoside.

In one embodiment, the phenol derivative and the phenol derivativeglycoside may be derived from a phenol derivative glycosylation reactionsolution.

In one embodiment, the glycosylation reaction solution may be ahesperidin or hydroquinone glycosylation reaction solution.

In one embodiment, the first aqueous solution may further contain aphase separation assisting agent.

In one embodiment, the glycosylation reaction solution may be ahesperidin or hydroquinone glycosylation reaction solution.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing an influence of glucose concentration ontetrahydrofuran extraction.

FIG. 2 is a graph showing the transfer rates of hydroquinone glycosideand glucose to a tetrahydrofuran phase.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail. As usedherein, concentration is represented by grams per 100 cubic centimetersof solution unless otherwise described. For example, “10% sodiumchloride solution” refers to a sodium chloride solution in which 10 g ofsodium chloride is dissolved per 100 cubic centimeters of solution.

A method according to the present invention is a method of extracting ahydrophobic group-containing water-soluble organic compound. The methodof the present invention comprises the step of contacting an aqueoussolution containing a hydrophobic group-containing water-soluble organiccompound and a saccharide with a polar organic solvent to obtain anaqueous phase and an organic phase so that the hydrophobicgroup-containing water-soluble organic compound is transferred to theorganic phase.

(1) Hydrophobic Group-containing Water-soluble Organic Compound

As used herein, “hydrophobic group-containing water-soluble organiccompound” refers to an organic compound which contains a hydrophobicgroup and is soluble in water.

As used herein, “water-soluble” compound refers to a compound that atleast 0.01 g can be dissolved in one liter of water at 20° C.Preferably, at least 0.1 g, more preferably at least 1 g, even morepreferably at least 5 g, and most preferably at least 10 g, of ahydrophobic group-containing water-soluble organic compound can bedissolved in one liter of water at 20° C. There is no particular upperlimit to the solubility, although the solubility is preferably no morethan 300 g in one liter of water at 20° C. More preferably, thesolubility is no more than 100 g in one liter of water at 20° C.

A hydrophobic group is preferably a hydrophobic group containing atleast three carbon atoms, and more preferably an aromatic residue.Examples of hydrophobic group-containing water-soluble organic compoundsinclude flavonoids, isoflavones, phenolic compounds, flavonoidglycosides, isoflavone glycosides, phenolic compound glycosides,hydroquinone glycosides, anthracene glycosides, water-soluble aromaticcompounds (e.g., chalcone glycosides), terpene glycosides, steroidglycosides, triterpenoid glycosides, alkaloid glycosides, andC-glycosides. Preferably, the hydrophobic group-containing water-solubleorganic compound is a water-soluble aromatic compound.

As used herein, “water-soluble aromatic compound” refers to a compoundwhich is soluble in water and has an aromatic group.

The water-soluble aromatic compound is preferably selected from thegroup consisting of phenol derivatives and glycosides thereof.

“Phenol derivative” refers to a compound having a phenol backbone (i.e.,a benzene ring) or a flavonoid backbone and having a hydroxyl grouplinked to the phenol backbone or the flavonoid backbone, includingphenol and kojic acid. Examples of the phenol derivative include acompound having a phenolic hydroxyl group on a single phenol orflavonoid backbone, and a compound having at least two phenolic hydroxylgroups on a single phenol or flavonoid backbone. Hereinafter, for thesake of convenience, compounds having one phenolic hydroxyl group on asingle phenol or flavonoid backbone are called monophenol typecompounds, and compounds having at least two phenolic hydroxyl groups ona single phenol or flavonoid backbone are called polyphenol typecompounds.

Compounds having two phenolic hydroxyl groups on a single phenol orflavonoid backbone are called diphenol compounds.

A phenol derivative glycoside having a phenolic hydroxyl group is alsoincluded as a phenol derivative.

Examples of monophenol type compounds having one phenolic hydroxyl groupon a single phenol or flavonoid backbone include phenol, salicylalcohol, kojic acid, dimethoxy phenol, acetaminophen, vanillin, anddaidzein.

Examples of monophenol compounds also include monophenol type flavonoidtype compounds. Examples of monophenol type flavonoid type compoundsinclude monophenol type flavone type compounds, monophenol typeisoflavone type compounds, monophenol type flavonol type compounds,monophenol type flavanone type compounds, monophenol type flavanonoltype compounds, monophenol type catechin type compounds, monophenol typeaurone type compounds, monophenol type chalcone type compounds, andmonophenol type dihydrochalcone type compounds.

Examples of dimethoxyphenols include 2,3-dimethoxy phenol, 2,4-dimethoxyphenol, 2,5-dimethoxy phenol, 2,6-dimethoxy phenol, 3,4-dimethoxyphenol, and 3,5-dimethoxy phenol. 3,4-dimethoxy phenol and 3,5-dimethoxyphenol are preferable.

Examples of polyphenol type compounds having at least two phenolichydroxyl groups on a single phenol or flavonoid backbone includehydroquinone, hesperetin, epigallocatechin, epicatechin gallate,anthocyanidin type compounds, anthocyanin type compounds, caffeic acid,catechol, resorcinol, protocatechuic acid, gallic acid, genistein,β-resorcylic acid, and phloroglucinol.

Examples of diphenol compounds also include diphenol type flavonoid typecompounds. Examples of diphenol type flavonoid type compounds includediphenol type flavone type compounds, diphenol type isoflavone typecompounds, diphenol type flavonol type compounds, diphenol typeflavanone type compounds, diphenol type flavanonol type compounds,diphenol type catechin type compounds, diphenol type aurone typecompounds, diphenol type chalcone type compounds, and diphenol typedihydrochalcone type compounds.

Examples of resorcylic acids include α-resorcylic acid, β-resorcylicacid, and γ-resorcylic acid. In the present invention, β-resorcylic acidis preferable.

As used herein, a “phenol derivative glycoside” is a substance in whicha phenol derivative moiety is linked to one or more saccharide moietywith glycoside linkage(s). The phenol derivative glycoside may be amono-glucopyranoside (e.g., hydroquinone-O-α-D-glucopyranoside, salicin,caffeic acid-O-α-D-glucopyranoside, 3,4-dimethoxyphenol-O-α-D-glucopyranoside, and catechin-O-α-D-glucopyranoside), adiglucopyranoside in which a saccharide moiety is additionally linked tothe above-described mono-glucopyranoside (e.g., a hesperidinderivative), a triglucopyranoside, and the like.

As used herein, a “glycoside” is a substance in which an aglycon islinked to one or more saccharide moieties with glycoside linkage(s). Thepolymerization degree of the saccharide moiety is preferably 1-10, morepreferably 1-5, and even more preferably 1-3. The saccharide moiety canbe a monosaccharide moiety or a disaccharide moiety. As used herein,glucosides are included in the definition of the glycoside. Glucosidesare glycosides in which one or more glucose moieties are linked to anaglycon.

The hydrophobic group-containing water-soluble organic compound ispreferably selected from the group consisting of salicin, coniferin,arbutin, sennoside, stevioside, rubsoside, rutin, hesperidin, naringin,daidzein, genistin, barbaroin, vanillin, saponins, berberine,kaempferol, baicalin, capillarin, catechin, corydaline, esculetin,epicatechin, gingerols, glycyrrhizin, Diosmin, neohesperidin, caffeicacid, salicyl alcohol, elladitannin, and hydroquinone. The hydrophobicgroup-containing water-soluble organic compound is more preferablyselected from the group consisting of hydroquinone glycoside, catechin,salicin, hesperidin, hesperidin glycosides, caffeic acid, salicylalcohol and elladitannin.

The hydrophobic group-containing water-soluble organic compound may bepresent in aqueous solution at any concentration. The concentration ofthe hydrophobic group-containing water-soluble organic compound ispreferably 0.01% to 50%, more preferably 0.1% to 40%, even morepreferably 0.5% to 30%, still even more preferably 1% to 20%, and mostpreferably 5% to 15%. If the concentration of the hydrophobicgroup-containing water-soluble organic compound present in aqueoussolution is excessively low, the purification efficiency may be poor. Ifthe concentration of the hydrophobic group-containing water-solubleorganic compound present in aqueous solution is excessively high, thehydrophobic group-containing water-soluble organic compound mayprecipitate. A concentration at which the hydrophobic group-containingwater-soluble organic compound does not precipitate is preferable.

The hydrophobic group-containing water-soluble organic compound may bederived from an enzyme reaction solution containing the hydrophobicgroup-containing water-soluble organic compound. As used herein, anenzyme reaction solution refers to a solution obtained by subjecting anystarting material to an enzyme reaction. Examples of such an enzymereaction solution include a glycosylation reaction solution, ahydrolysis reaction solution, a transfer reaction, and a condensationreaction solution for the hydrophobic group-containing water-solubleorganic compound. The enzyme reaction solution preferably contains asaccharide. The enzyme reaction solution is typically a reactionsolution after a reaction has proceeded and a reaction product has beenproduced.

An example of a glycosylation reaction is representatively a glycosyltransfer reaction for a glycosyl transfer acceptor which is catalyzed bycyclodextrin glucanotransferase. Examples of such a glycosyl transferacceptor include a flavonoid containing a saccharide in the structurethereof, a flavonoid not containing a saccharide in the structurethereof, a phenol compound, and a phenolic compound glycoside. Examplesof a representative glycosyl transfer acceptor include hesperidin,naringin, neohesperidin, and rutin.

Another example of the glycosylation reaction is a glycosyl transferreaction for a glycosyl transfer acceptor, 5 which is catalyzed by atransferring type amylase. Examples of such a glycosyl transfer acceptorinclude catechin, caffeic acid, kojic acid, hydroquinone, catechol,resorcinol, protocatechuic acid, gallic acid, vanillin, daidzein,genistein, α-resorcylic acid and phloroglucinol.

The glycosylation reaction solution is preferably a glycosylationreaction solution for hesperidin or hydroquinone.

Examples of an enzyme catalyzing an enzyme reaction include, in additionto cyclodextrin glucanotransferase and transferring type amylase,α-amylase, pullulanase, amylomaltase, D-enzyme, neopullulanase,cyclodextrinase, α-glucosidase, cellulase, β-glucosidase, andβ-galactosidase.

The enzyme reaction solution containing the hydrophobic group-containingwater-soluble organic compound may be designed and obtained by a methodknown to those skilled in the art.

The hydrophobic group-containing water-soluble organic compound may alsobe derived from any natural material containing the hydrophobicgroup-containing water-soluble organic compound. The hydrophobicgroup-containing water-soluble organic compound may be derived from, forexample, an organism (e.g., an animal or a plant). Alternatively, ananimal extract or a plant extract can be used. The animal extract refersto any substance extracted from an animal. The plant extract refers toany substance extracted from a plant. For example, any hydrophobicgroup-containing water-soluble organic compound obtained from a leaf,stem, root, flower, fruit, or the like of a plant can be used. Examplesof a plant material include soybean, processed soybean products,sea-cucumber, Chinese nutgall, Scutellaria root (Scutellariae Radix),aloe, Rehmannia root (Rehmanniae Radix), Asiatic ginseng (Panaxginseng), Peony root (Paeoniae Radix), Gardenia jasminoides, Glycyrrhiza(Glycyrrhizae Radix), Bupleurum root (Bupleuri Radix), Rhubarb (Rheirhizome), Houttuyniacordata, Cowberry (Vaccinium vitis-idaea), tea,Sweet tea (Chinese blackberry tea; Rubus suanissm S.Lee), and citrus(e.g., the fruit of orange). Any hydrophobic group-containingwater-soluble organic compound present in the body of an animal can beused.

(2) Saccharides

As used herein, saccharides refer to compounds having the generalformula C_(n)(H₂O)_(m). Saccharides are grouped into monosaccharides,oligosaccharides, and polysaccharides according to the number ofconstituents, i.e., saccharide units. In the present invention,monosaccharides and oligosaccharides are preferable. In the presentinvention, a saccharide, which is soluble in water or, if not, has waterholding capacity, is preferable.

Examples of monosaccharides include D-glucose, galactose, fructose,arabinose, xylose, and rhamnose. A preferable monosaccharide isD-glucose.

Oligosaccharides as used herein refer to substances obtained bydehydration-condensation of 2 to 10 monosaccharides. An oligosaccharidehas preferably 2 to 9 saccharide units, more preferably 2 to 8saccharide units, and even more preferably 2 to 7 saccharide units.Examples of oligosaccharide include sucrose, lactose,malto-oligosaccharides, galacto-oligosaccharides,lacto-oligosaccharides, and fructo-oligosaccharides. Examples of themalto-oligosaccharides include maltose, maltotriose, maltotetraose,maltopentaose, and maltohexaose, maltoheptaose, malto-octaose,maltononaose, and maltodecaose. An oligosaccharide may be astraight-chain oligosaccharide or a branched-chain oligosaccharide. Anoligosaccharide may have an intramolecular ring structure.

Polysaccharides as used herein refer to substances generated bydehydration-condensation of at least 11 monosaccharides. Apolysaccharide preferably has at least one α-1,4 linkage. Examples ofpolysaccharides include dextrin, amylose, amylopectin, starch, dextran,and cellulose.

Dextrins refer to substances obtained by lowering the molecular weightof starch by a chemical or enzymatic method. Examples of dextrinsinclude British gum, yellow dextrin, white dextrin, PINE-DEX (MatsutaniChemical Industry Co., Ltd.), SUNDECK (Sanwa Cornstarch Co., Ltd.), andTetrup (Hayashibara Shoji, Inc.).

Amylose is a straight-chain molecule composed of glucose units linkedtogether by α-1,4 linkages. Amylose is contained in natural starch.

Amylopectin is a branched-chain molecule composed of glucose unitslinked together by α-1,4 linkages to which glucose units are linkedtogether by α-1,6 linkages. Amylopectin is contained in natural starch.As amylopectin, for example, waxy corn starch consisting of 100%amylopectin may be used.

Starch is a mixture of amylose and amylopectin. As starch, any starchwhich is usually commercially available may be used. The ratio ofamylose to amylopectin contained in starch varies depending on the typeof plant which produces the starch. The majority of starch contained inwaxy rice, waxy corn, and the like is amylopectin. Starch is dividedinto natural starch, starch degradation products, and processed starch.

Natural starch is divided into tuber starch and cereal starch accordingto a raw material from which it is derived. Examples of tuber starchinclude potato starch, tapioca starch, sweet potato starch, kudzustarch, bracken starch, and the like. Examples of cereal starch includecorn starch, wheat starch, rice starch, and the like.

Processed starch is starch obtained by subjecting natural starch totreatment, such as hydrolysis, esterification, gelatinization, or thelike, to confer properties for better ease of utilization. A widevariety of processed starches are available which have variouscombinations of properties, such as, for example, temperature at whichgelatinization starts, the viscosity of the starch paste, thetransparency of the starch paste, aging stability, and the like. Thereare various types of processed starch. An example of such starch isstarch which is obtained by immersing starch granules in acid at atemperature of no more than the gelatinization temperature of the starchso that starch molecules are cleaved but starch granules are not broken.

Starch degradation products are oligosaccharides or polysaccharidesobtained by subjecting starch to treatment, such as enzyme treatment,hydrolysis, or the like, which have a lower molecular weight than beforethe treatment. Examples of the starch degradation products includestarch degraded by a debranching enzyme, starch degraded byphosphorylase, and starch partially degraded by hydrolysis.

Starch degraded by a debranching enzyme is obtained by allowing adebranching enzyme to act on starch. By changing the action time of thedebranching enzyme to various extents, starch degraded by a debranchingenzyme in which branching portions (i.e., α-1,6-glucoside linkage) arecleaved to any extent can be obtained. Examples of the starch degradedby a debranching enzyme include degradation products of 4 to 10,000saccharide units having 1 to 20α-1,6-glucoside linkages, degradationproducts of 3 to 500 saccharide units without any α-1,6-glucosidelinkages, malto-oligosaccharide, and amylose. In the case of starchdegraded by a debranching enzyme, the distribution of the molecularweight of the resultant degradation products may vary depending on thetype of starch to be degraded. The starch degraded by a debranchingenzyme may be a mixture of saccharide chains having various lengths.

Dextrin and starch partially degraded by hydrolysis refer to degradationproducts obtained by degrading starch partially by the action of anacid, an alkali, an enzyme, or the like. In the present invention, thenumber of saccharide units contained in dextrin and starch partiallydegraded by hydrolysis is preferably about 10 to about 100,000, morepreferably about 50 to about 50,000, and even more preferably about 100to about 10,000. In the case of dextrin and starch partially degraded byhydrolysis, the distribution of the molecular weight of the resultantdegradation products may vary depending on the type of starch to bedegraded.

Dextrin and the starch partially degraded by hydrolysis may be a mixtureof saccharide chains having various lengths.

Dextran refers to α-1,6-glucan.

Cellulose is a straight-chain molecule composed of glucose units linkedtogether by β-1,4-glucoside linkages.

The saccharide may be a single compound or a mixture of a plurality ofcompounds.

The saccharide may be originally contained in an aqueous solutioncontaining the hydrophobic group-containing water-soluble organiccompound, or may be added to an aqueous solution containing thehydrophobic group-containing water-soluble organic compound. Thesaccharide is preferably originally contained in an aqueous solutioncontaining the hydrophobic group-containing water-soluble organiccompound. Examples of such an aqueous solution include theabove-described glycosylation reaction solutions and fruit juice.

The saccharide preferably is a small molecule. When a solution containsa relatively high molecular weight polysaccharide, as does aglycosylation reaction solution, an enzyme cleaving saccharide chains,such as glucoamylase, may be added to the solution which is allowed toreact, thereby degrading the polysaccharide to monosaccharides oroligosaccharides. It is preferable that a polysaccharide in an aqueoussolution is degraded to monosaccharides or oligosaccharides before beingbrought into contact with an organic solvent. It is preferable that allsaccharides are dissolved in an aqueous solution. However, saccharidemay be partially suspended in an aqueous solution as long as thesaccharide does not interfere with separation of the aqueous solutionfrom an organic solvent.

The saccharide concentration of an aqueous solution may be anyconcentration. The saccharide concentration of an aqueous solution ispreferably at least 12%, more preferably at least 20%, even morepreferably at least 30%, still even more preferably at least 40%, andmost preferably at least 50%. The upper limit of the saccharideconcentration is any concentration as long as the saccharide and thehydrophobic group-containing water-soluble organic compound do notprecipitate. For example, the upper limit is no more than 90%, no morethan 80%, no more than 70%, no more than 60%, or no more than 55%.

The saccharide concentration of an aqueous solution may be measured by amethod known in the art. For example, as a simple method, there is ameasuring method using a Brix scale. The measuring method using a Brixscale is simple, but cannot measure each of saccharide separately. Inorder to measure each of the saccharides separately, for example, anaqueous solution containing saccharides may be subjected to HPLC using acolumn LiChrosorb NH₂ (manufactured by Merck; 4.0×250 mm) and using amixture of water and acetonitrile at 25:75 (v/v) as a mobile phase, andthe eluate may be measured using an RI detector.

The pH of an aqueous solution is preferably 2 to 11, more preferably 3to 9, and even more preferably 4 to 8.

(3) Polar Organic Solvent

As used herein, “polar organic solvent” refers to an organic solventwhich has a solvent strength (ε⁰) to alumina of at least 0.4 and ismiscible with distilled water. The solvent strength of a polar organicsolvent is preferably 0.42 to 0.98, more preferably 0.44 to 0.95, andmost preferably 0.44 to 0.90. A polar organic solvent may be an organicsolvent having a permittivity of at least 7.0 at 20° C. A polar organicsolvent preferably has a permittivity of 7.3 to 40.0, more preferably7.4 to 39.0, and most preferably 7.5 to 38.0 at 20° C.

Examples of the solvent strength to alumina are shown in Table 1.Examples of the permittivity are shown in Table 2. Examples of polarorganic solvents used in the present invention include polar organicsolvents shown in the following Tables 1 and 2, which have a solventstrength or a permittivity within the above-described ranges.

TABLE 1 Solvent strength Solvent to alumina (ε⁰) fluoroalkane −0.25  n-pentane 0.00 isooctane 0.01 hexane 0.01 n-decane 0.04 cyclohexane 0.04cyclopentane 0.05 carbon disulfide 0.15 carbon tetrachloride 0.18 xylene0.26 isopropyl ether 0.28 toluene 0.29 benzene 0.32 ethyl ether 0.38chloroform 0.40 methylene chloride 0.42 methyl isobutyl ketone 0.43tetrahydrofuran 0.45 ethylene dichloride 0.49 methyl ethyl ketone 0.51acetone 0.56 dioxane 0.56 ethyl acetate 0.58 methyl acetate 0.60dimethyl suffixed 0.62 aniline 0.62 diethyl amine 0.63 nitromethane 0.64acetonitrile 0.65 pyridine 0.71 isopropanol 0.82 n-propanol 0.82 ethanol0.88 methanol 0.95 ethylene glycol 1.11 acetic acid great water great

TABLE 2 Solvent e isooctane 1.94 n-hexane 1.88 n-heptane 1.92 diethylether 4.33 cyclohexane 2.02 ethyl acetate 6.02 toluene 2.38 chloroform4.81 tetrahydrofuran 7.58 benzene 2.27 acetone 20.7 dichloromethane 8.93dioxane 2.25 propanol 20.33 ethanol 25.8 dimethylformamide 36.7acetonitrile 37.5 acetic acid 6.3 dimethyl sulfoxide 4.7 methanol 32.7water 81.1 e: permittivity

A polar organic solvent is preferably tetrahydrofuran, isopropanol,acetonitrile, acetone, ethanol, methanol, propanol, pyridine, ordimethoxy sulfoxide, more preferably tetrahydrofuran, isopropanol,acetonitrile or acetone, and most preferably tetrahydrofuran oracetonitrile. When an aqueous solution further contains a phaseseparation assisting agent, a polar organic solvent is preferablytetrahydrofuran, acetonitrile, acetone or isopropyl alcohol, and morepreferably acetone or isopropyl alcohol.

A polar organic solvent is preferably a single compound. However, amixture of at least two polar organic solvents may be used as long as anorganic phase is not separated into two phases. A polar organic solvent,which is appropriate for extraction of a hydrophobic group-containingwater-soluble organic compound from an aqueous solution, may be selectedby those skilled in the art as required.

The amount of a polar organic solvent which is brought into contact withan aqueous solution is representatively 0.1 to 10 times, and morepreferably 0.2 to 2 times the volume of an aqueous solution.

(4) Phase Separation Assisting Agent

An aqueous solution may contain a phase separation assisting agent. Asused herein, “phase separation assisting agent” refers to a substancewhich assists separation of a mixture of an aqueous solution and a polarorganic solvent into an aqueous phase and an organic phase. Note that asaccharide and a hydrophobic group-containing water-soluble organiccompound are not phase separation assisting agents, even if they have anaction of assisting phase separation. A phase separation assisting agentmay be a salt having a salting-out effect and a water-soluble substancecapable of enhancing ionic strength. A phase separation assisting agentmay be a salt or an organic acid. Examples of phase separation assistingagents include, but are not limited to, sulfates (e.g., ammonium sulfateand magnesium sulfate), sodium salts (e.g., sodium chloride and sodiumsulfite), phosphates (e.g., potassium phosphate, sodium phosphate,magnesium phosphate, and ammonium phosphate), acetates (e.g., sodiumacetate and potassium acetate), lactates (e.g., sodium lactate andmagnesium lactate), organic acids (e.g., citric acid, sodium citrate,ascorbic acid, sodium ascorbate, and malic acid), and ammonium chloride.A phase separation assisting agent is preferably a salt and is morepreferably selected from the group consisting of sodium chloride, sodiumcitrate, magnesium sulfate and ammonium sulfate.

A phase separation assisting agent may be contained in an aqueoussolution in a sufficient amount to assist phase separation. Such anamount is known to those skilled in the art. The concentration of aphase separation assisting agent contained in an aqueous solution ispreferably at least 5%, more preferably 10%, even more preferably atleast 15%, and most preferably at least 20%. There is no particularupper limit to the amount of a phase separation assisting agent,although it is preferably no more than 50% and more preferably no morethan 40%.

It is preferable that a phase separation assisting agent is contained inan aqueous solution in advance. However, a phase separation assistingagent can be added while an aqueous solution and a polar organic solventare brought into contact with each other.

(Preparation of an Aqueous Solution Containing a HydrophobicGroup-containing Water-soluble Organic Compound and a Saccharide)

An aqueous solution containing a hydrophobic group-containingwater-soluble organic compound and a saccharide may be prepared by amethod known to those skilled in the art. Such an aqueous solution maybe an enzyme reaction solution which is not subjected to any treatmentafter the enzyme reaction has proceeded, or an enzyme reaction solutionwhich is concentrated, diluted, filtered, or pH-adjusted after theenzyme reaction has proceeded. Particularly, when the viscosity of anenzyme reaction solution is too high to be stirred, it is preferable todilute the enzyme reaction solution. Alternatively, an aqueous solutionmay be prepared by adding a phase separation assisting agent to anenzyme reaction solution containing a hydrophobic group-containingwater-soluble organic compound and a saccharide, or a concentratedsolution thereof.

An aqueous solution may also be an extract of an organism selected fromanimals or plants, which contains a hydrophobic group-containingwater-soluble organic compound. Such an extract may be prepared byextracting an animal material or a plant material containing ahydrophobic group-containing water-soluble organic compound by a methodknown in the art. An exemplary extraction method comprises: providing ananimal material or a plant material containing a hydrophobicgroup-containing water-soluble organic compound into an extractionsolvent, such as water (e.g., water having a temperature of more than 0°C. and less than 40° C.), warm water (e.g., water having a temperatureof no less than 40° C. and less than 60° C.), hot water (e.g., waterhaving a temperature of no less than 60° C. and less than 100° C.),alcohol, pyridine, ethyl acetate, or a mixture thereof; allowing thehydrophobic group-containing water-soluble organic compound to betransferred from the animal material or the plant material to theextraction solvent; removing the animal material and the plant materialfrom the extraction solvent to obtain an extract solution; andconcentrating or drying the extract solution if necessary. It ispreferable that the extract does not contain an organic solvent. Whenthe extraction solvent is an organic solvent, it is preferable to removethe organic solvent by concentrating the extract solution. The extractmay be liquid or solid. Juice obtained by squeezing an animal materialor a plant material is also herein included in the definition of anextract. An aqueous solution is preferably fruit juice. An animalmaterial may be the entire animal or any organ or tissue of an animal. Aplant material may be the entire plant or any organ (e.g., flower,fruit, seed, root, stem, and leaf) or tissue of a plant. An animalmaterial and a plant material to be subjected to extraction may beeither raw or dried. If an extract is an aqueous solution, the extractmay be used as it is in the present invention. An aqueous solution maybe prepared from an extract by concentration, dilution, or the like.Particularly, when the viscosity of an extract is too high to bestirred, it is preferable to dilute the extract. Note that as long as ahydrophobic group-containing water-soluble organic compound of interestis dissolved in an aqueous solution to be used in the method of thepresent invention, any other ingredient may be suspended therein.Alternatively, an aqueous solution may be prepared by adding a phaseseparation assisting agent to an animal extract containing a hydrophobicgroup-containing water-soluble organic compound and a saccharide, aplant extract containing a hydrophobic group-containing water-solubleorganic compound and a saccharide, or a concentrate or diluent of theseextracts. For example, an aqueous solution may be prepared by adding aphase separation assisting agent to fruit juice or a concentratethereof.

An effective ingredient selected from the group consisting of salicin,coniferin, arbutin, sennoside, stevioside, rubsoside, rutin, hesperidin,naringin, daidzein, genistin, barbaroin, vanillin, saponins, berberine,kaempferol, baicalin, capillarin, catechin, corydaline, esculetin,epicatechin, gingerols and glycyrrhizin, is preferably dissolved in anextract solution.

(Extraction of a Hydrophobic Group-containing Water-soluble OrganicCompound)

In a method according to the present invention, an aqueous solutioncontaining a hydrophobic group-containing water-soluble organic compoundand a saccharide is brought into contact with a polar organic solvent toobtain an aqueous phase and an organic phase, thereby the hydrophobicgroup-containing water-soluble organic compound is allowed to betransferred to the organic phase.

An aqueous solution and a polar organic solvent may be brought intocontact with each other by, for example, mixing the aqueous solution andthe polar organic solvent. Bringing an aqueous solution and a polarorganic solvent into contact with each other is also called extraction.A temperature at which an aqueous solution and a polar organic solventare brought into contact with each other is preferably 10° C. to 50° C.,more preferably 25° C. to 45° C., even more preferably 20° C. to 40° C.,and most preferably 25° C. to 35° C.

An aqueous solution and a polar organic solvent are mixed and stirred,followed by allowing them to stand, resulting in separation to anaqueous phase and an organic phase. In general, the aqueous phase andthe organic phase form respective layers, i.e., a water layer and anorganic layer. In general, the solvent layer, which has a greaterspecific gravity, is the lower layer. When an organic solvent which hasa smaller specific gravity than that of water is used, the water layertypically is the lower layer while the upper layer is the organic layer.

Typically, an aqueous phase contains a small amount of polar organicsolvent, while an organic phase contains a small amount of water. Forexample, acetone is added to an aqueous solution or a suspensioncontaining a hydrophobic group-containing water-soluble organic compound(e.g., hesperidin and rutin), which are in turn stirred and then allowedto stand. When they are separated into an aqueous phase and an organicphase (acetone phase), a small amount of water is dissolved in theacetone phase. Therefore, compared with the case when water is notcontained in the organic phase, the solubility of the hydrophobicgroup-containing water-soluble organic compound increases, thehydrophobic group-containing water-soluble organic compound isefficiently dissolved in acetone.

When an aqueous solution and a polar organic solvent are mixed tocontact each other, the mixture is preferably stirred. Examples of amethod for stirring include, but are not limited to, rotating, shaking,and both. In order to efficiently extract and purify a hydrophobicgroup-containing water-soluble organic compound, a multistagecounterflow partition apparatus (also called a continuous liquid-liquidextraction apparatus) can be used.

(Purification of a Phenol Derivative Glycoside)

The method of the present invention is particularly useful forpurification of a phenol derivative glycoside. Purification of a phenolderivative glycoside will be described as an example in more detail.

A phenol derivative glycoside may be formed by, for example, causing asaccharide (e.g., a malto-oligosaccharide or starch) to react with aphenol derivative in the presence of an enzyme. Typically, this reactionreaches equilibrium at a certain level and does not proceed further.Therefore, in this enzyme reaction solution, the phenol derivative, thephenol derivative glycoside and the saccharide are present. When theenzyme reaction solution contains a polysaccharide or anoligosaccharide, a glycolytic enzyme, such as glucoamylase, may be addedto the enzyme reaction solution, followed by incubation, to degrade thepolysaccharide or the oligosaccharide in the enzyme reaction solution tomonosaccharides. The degradation of the polysaccharide or theoligosaccharide to monosaccharides increases the mole value, i.e., molarconcentration, without changing the total weight of the saccharides,resulting in promotion of separation of an aqueous phase and an organicphase. When the enzyme reaction solution and the polar organic solventare brought into contact with each other to obtain a first aqueous phaseand an organic phase containing a small amount of water, the phenolderivative and the phenol derivative glycoside are transferred to theorganic phase. As described above, in order to promote phase separation,a phase separation assisting agent may be added to the aqueous phase toobtain an aqueous solution containing the phase separation assistingagent, and then the aqueous solution may be brought into contact withthe polar organic solvent.

Thereafter, the organic phase containing a small amount of water isrecovered. A phenol derivative and a phenol derivative glycoside containa hydrophobic portion which causes them to have a higher affinity to anorganic phase than that of a saccharide. Therefore, the phenolderivative and the phenol derivative glycoside are efficientlytransferred to the organic phase, the saccharide is less transferred tothe organic phase. Therefore, the phenol derivative and the phenolderivative glycoside are extracted in the recovered organic phase. Ingeneral, assuming that the same total amount of a polar organic solventis used for partition extraction, if the polar organic solvent isdivided into some aliquots and the aliquots are used to performextraction from an aqueous solution a plurality of times, the efficiencyof the extraction is greater than when the whole amount of the polarorganic solvent is used a single time to contact the aqueous solutionfor extraction. Therefore, a step of bringing an aqueous phase remainingafter an organic phase is recovered into contact with a polar organicsolvent to obtain an aqueous phase and an organic phase containing asmall amount of water again and recovering the organic phase, may beperformed twice or more. The recovery operation of an organic phase isperformed at least twice, the obtained organic phases may be combinedtogether and may be used in a subsequent step.

Thereafter, the polar organic solvent is removed from the organic phasecontaining a small amount of water. A method for removing the polarorganic solvent from the organic phase may be any method known to thoseskilled in the art. Examples of such a method include concentrationusing an evaporator and evapor. The polar organic solvent may becompletely removed, or may remain in a small amount as long as it doesnot interfere with a subsequent step. After the removal of the polarorganic solvent, the phenol derivative and the phenol derivativeglycoside remain in a small amount of water which has been contained inthe polar organic solvent. In this removal step, it is preferable toavoid much water from being removed. In this removal step, it ispreferable that the phenol derivative and the phenol derivativeglycoside do not precipitate. If the phenol derivative and the phenolderivative glycoside precipitate, it is preferable that the phenolderivative and the phenol derivative glycoside are dissolved by additionof water. Thus, a second aqueous solution containing the phenolderivative and the phenol derivative glycoside is obtained.

Thereafter, the second aqueous solution is brought into contact withethyl acetate to obtain a second aqueous phase and an ethyl acetatephase, so that the phenol derivative is transferred to the ethyl acetatephase.

Thereafter, the second aqueous phase is recovered. Since the phenolderivative does not contain a glycoside moiety, the phenol derivativehas a higher affinity to the ethyl acetate phase than that of the phenolderivative glycoside. Therefore, the phenol derivative is efficientlytransferred to the ethyl acetate phase, while the phenol derivativeglycoside is less transferred to the ethyl acetate phase. Therefore, thephenol derivative glycoside remains in the recovered aqueous phase. Whena small amount of saccharide remains in the second solution, thesaccharide remains in the aqueous phase. As in the step of contactingthe aqueous solution and the polar organic solvent and the step ofrecovering the organic phase, a step of bringing the aqueous phaseremaining after the removal of the ethyl acetate phase into contact withethyl acetate to obtain an aqueous phase and an ethyl acetate phaseagain and recovering the second aqueous phase may be performed at leasttwice.

Note that although a method is herein described in which the step ofcontacting an organic phase with ethyl acetate is performed after thestep of contacting the aqueous solution and the polar organic solventand the step of recovering the organic phase, the aqueous solution maybe brought into contact with ethyl acetate and an aqueous phase isrecovered before the aqueous phase is brought into contact with a polarorganic solvent.

Thereafter, the second aqueous phase is concentrated and cooled so as toprecipitate the phenol derivative glycoside. It is preferable toconcentrate the second aqueous phase so that the phenol derivativeglycoside in the second aqueous phase reaches a concentration of atleast 10%, preferably at least 15%, more preferably at least 20%, andmost preferably at least 25%. This is done by utilizing the fact thatthe saturation concentration in water of a phenol derivative glycosideis lower than the saturation concentration in water of a saccharide. Forexample, assuming that the saccharide is glucose and the phenolderivative glycoside is hydroquinone glycoside, the fact that thesaturation concentration in water of glucose is about 18% and thesaturation concentration in water of hydroquinone glycoside is about 10%is utilized.

EXAMPLES

Next, the present invention will be described in more detail by way ofexamples, although the present invention is not limited to the examples.

Experimental Example 1 Influence of a Saccharide on Phase Separation

Glucose, sucrose and fructose were used as representatives of respectivetypes of saccharide so that an influence thereof on phase separation ofan aqueous solution and a polar organic solvent could be studied.

Specifically, glucose, sucrose or fructose was dissolved in water toprepare 10%, 15%, 20%, 25%, 30% and 50% aqueous solutions. 10 ml ofacetonitrile or tetrahydrofuran was added to 10 ml of each aqueoussaccharide solution, followed by vigorous stirring using a separatingfunnel for 5 minutes. Both acetonitrile and tetrahydrofuran are polarorganic solvents which are water miscible and when they are mixed withwater, the mixture does not undergo separation into an aqueous phase andan organic phase. After the stirring, the mixture was allowed to standfor 30 minutes, observing whether or not the mixture was separated intotwo phases, an aqueous phase and an organic phase. The results from theaddition of tetrahydrofuran or acetonitrile to each aqueous solution areshown in Tables 3 to 5. In the tables, THF represents tetrahydrofuranand AcCN represents acetonitrile.

TABLE 3 Glucose 50% 30% 25% 20% 15% 10% AcCN Yes Yes Yes Yes Yes No THFYes Yes Yes No No No Yes: Separated No: Not separated

TABLE 4 Sucrose 50% 30% 25% 20% 15% 10% AcCN Yes Yes Yes Yes Yes No THFYes No No No No No Yes: Separated No: Not separated

TABLE 5 Fructose 50% 30% 25% 20% 15% 10% AcCN Yes Yes Yes Yes Yes No THFYes Yes No No No No Yes: Separated No: Not separated

As shown in Tables 3 to 5, it was found that when acetonitrile ortetrahydrofuran was mixed with the aqueous saccharide solution, when acertain amount of saccharide was present, phase separation occurred.

According to the results, it was found that adjustment of the type andconcentration of a saccharide contained in an aqueous solution wouldfacilitate phase separation and, for example, it is preferable to adjusta saccharide concentration of at least about 12%.

Experimental Example 2 Influence of a Salt on Phase Separation

Next, an influence of a salt on phase separation was studied.

Specifically, sodium chloride was added to an aqueous glucose solutionhaving a glucose concentration of 25% or 50% to a concentration of 20%or 10%, respectively, to prepare a sodium chloride-containing aqueousglucose solution. 10 ml of acetone or isopropanol, which is a polarorganic solvent, was added to 10 ml of each aqueous solution, followedby vigorous stirring using a separating funnel 5 minutes. Afterstirring, the mixture was allowed to stand for 30 minutes, observingwhether the mixture was separated into two phases. The results are shownin Table 6.

TABLE 6 Glucose 50% + Glucose 25% + NaCl 20% NaCl 10% Acetone Yes No2-ProOH Yes Yes Yes: Separated No: Not separated

Sodium citrate was added to an aqueous solution having a dextrinconcentration of 25%, to a concentration of 20% to prepare a sodiumcitrate-containing aqueous dextrin solution. Note that dextrin is aPINE-DEX #1 manufactured by Matsutani Chemical Industry Co., Ltd., whichhas DE of 7 to 9. As used herein, “DE” is an index which shows thedegree of degradation of a starch and is a percentage of direct reducingsaccharide converted to glucose in the solid content. Magnesium sulfatewas added to an aqueous solution having a glucose concentration of 25%to a concentration of 10% to prepare an aqueous magnesiumsulfate-containing glucose solution. 10 ml of acetone, which is a polarorganic solvent, was added to 10 ml of each aqueous solution, followedby vigorous stirring using a separating agent for 5 minutes. Afterstirring, the mixture was allowed to stand for 30 minutes, observingwhether the mixture was separated into two phases. The results are shownin Table 7.

TABLE 7 Dextrin 25% + Glucose 25% + Sodium Magnesium Citrate 20% Sulfate10% Acetone Yes Yes Yes: Separated No: Not separated

As shown in Tables 6 and 7, when the sodium chloride-containing aqueousglucose solution, the sodium citrate-containing aqueous dextrin solutionand the magnesium sulfate-containing aqueous glucose solution wererespectively mixed with acetone or isopropanol, phase separationoccurred. The addition of a salt, such as sodium chloride, sodiumcitrate, or magnesium sulfate, could lead to the occurrence of phaseseparation in a polar organic solvent which does not otherwise undergophase separation in the case of 50% aqueous saccharide solution. As aresult, it was found that a salt, such as sodium chloride, acts as aphase separation assisting agent. It is believed that a salt acts as aphase separation assisting agent by increasing the ionic strength of anaqueous solution in which the salt is dissolved. Therefore, anysubstance which can increase ionic strength is considered to be able tobe used as a phase separation assisting agent.

Example 1 Extraction of Catechins Example 1a

1 g of a mixture of catechins (SUNPHENON®; manufactured by Taiyo KagakuCo., Ltd.) was dissolved in 100 ml of an aqueous solution containing 30%glucose to obtain a sample aqueous solution. The absorbance at 280 nm ofthe sample aqueous solution was measured. 10 ml of tetrahydrofuran wasadded to 10 ml of the sample aqueous solution, followed by vigorousstirring using a separating funnel for 5 minutes. Thereafter, themixture was allowed to stand for 30 minutes. As a result, the mixturewas separated into two phases. The lower phase (aqueous phase) wasrecovered, and the volume and the absorbance at 280 nm thereof weremeasured. The amount of the catechins extracted in the upper phase(polar organic solvent phase) was calculated from a reduction in theabsorbance at 280 nm of the lower phase and the solution volume of thelower phase. As a result, it was found that by a single extractionoperation, 94.1% of the starting amount of catechins was extracted inthe tetrahydrofuran phase.

Example 1b

This additional extraction operation to Example 1a was conducted exceptfor use of 10 ml of acetonitrile instead of 10 ml of tetrahydrofuran. Asa result, it was found that by a single extraction operation, 84.4% ofthe starting amount of catechins was extracted in the acetonitrilephase.

Example 2 Extraction of Catechins Example 2a

1 g of a mixture of catechins (SUNPHENON®; manufactured by Taiyo Kagaku)was dissolved in 100 ml of an aqueous solution containing 30% glucoseand 10% sodium chloride to obtain a sample aqueous solution. Theabsorbance at 280 nm of the sample aqueous solution was measured. 10 mlof acetone was added to 10 ml of the sample aqueous solution, followedby vigorous stirring using a separating funnel for 5 minutes.Thereafter, the mixture was allowed to stand for 30 minutes. As aresult, the mixture was separated into two phases. The lower phase(aqueous phase) was recovered, and the volume and the absorbance at 280nm thereof were measured. The amount of the catechins extracted in theupper phase (polar organic solvent phase) was calculated from areduction in the absorbance at 280 nm of the lower phase and thesolution volume of the lower phase. As a result, it was found that by asingle extraction operation, 91.8% of the starting amount of catechinswas extracted in the acetone phase.

Example 2b

This additional extraction operation to Example 2a was conducted exceptfor use of 10 ml of isopropanol instead of 10 ml of acetone. As aresult, it was found that by a single extraction operation, 93.2% of thestarting material of catechins was extracted in the isopropanol phase.

Example 3 Extraction of Hesperidin Glycosides Example 3a

1 g of hesperidin glycosides (manufactured by Toyo Sugar Refining Co.,Ltd.) were dissolved in 100 ml of an aqueous solution containing 30%glucose to obtain a sample aqueous solution. The absorbance at 280 nm ofthe sample aqueous solution was measured. 10 ml of tetrahydrofuran wasadded to 10 ml of the sample aqueous solution, followed by vigorousstirring using a separating funnel for 5 minutes. Thereafter, themixture was allowed to stand for 30 minutes. As a result, the mixturewas separated into two phases. The lower phase (aqueous phase) wasrecovered, and the volume and the absorbance at 280 nm thereof weremeasured. The amount of hesperidin glycosides extracted in the upperphase (polar organic solvent phase) was calculated from a reduction inthe absorbance at 280 nm of the lower phase and the solution volume ofthe lower phase. As a result, it was found that by a single extractionoperation, 50.0% of the starting amount of hesperidin glycosides wasextracted in the tetrahydrofuran phase. Further, similar operations andmeasurements were performed twice more by adding 5 ml of tetrahydrofuranto the aqueous phase recovered after the tetrahydrofuran extraction. Asa result, it was found that a total of 80.0% of the hesperidinglycosides was extracted.

Example 3b

This additional extraction operation to Example 3a was conducted exceptfor use of 10 ml of acetonitrile instead of 10 ml of tetrahydrofuran. Asa result, it was found that by a single extraction operation, 27.6% ofthe starting amount of hesperidin glycosides was extracted in theacetonitrile phase.

Example 4 Extraction of Hesperidin Glycosides Example 4a

1 g of hesperidin glycosides (Toyo Sugar Refining Co., Ltd.) weredissolved in 100 ml of an aqueous solution containing 30% glucose and10% sodium chloride to obtain a sample aqueous solution. The absorbanceat 280 nm of the sample aqueous solution was measured. 10 ml of acetonewas added to 10 ml of the sample aqueous solution, followed by vigorousstirring using a separating funnel for 5 minutes. Thereafter, themixture was allowed to stand for 30 minutes. As a result, the mixturewas separated into two phases. The lower phase (aqueous phase) wasrecovered, and the volume and the absorbance at 280 nm thereof weremeasured. The amount of hesperidin glycosides extracted in the upperphase (polar organic solvent phase) was calculated from a reduction inthe absorbance at 280 nm of the lower phase and the solution volume ofthe lower phase. As a result, it was found that by a single extractionoperation, 43.5% of the starting amount of hesperidin glycosides wasextracted in the acetone phase. Further, similar operations andmeasurements were performed twice more by adding 5 ml of acetone to theaqueous phase recovered after the acetone extraction. As a result, itwas found that a total of 90.0% of the hesperidin glycosides wasextracted.

Example 4b

This additional extraction operation to Example 4a was conducted exceptfor use of 10 ml of isopropanol instead of 10 ml of acetone. As aresult, it was found that by a single extraction operation, 32.0% of thestarting amount of hesperidin glycosides was extracted in theisopropanol phase.

Example 5 Extraction of Salicin Example 5a

1 g of salicin was dissolved in 100 ml of an aqueous solution containing30% glucose to obtain a sample aqueous solution. The absorbance at 280nm of the sample aqueous solution was measured. 10 ml of tetrahydrofuranwas added to 10 ml of the sample aqueous solution, followed by vigorousstirring using a separating funnel for 5 minutes. Thereafter, themixture was allowed to stand for 30 minutes. As a result, the mixturewas separated into two phases. The lower phase (aqueous phase) wasrecovered, and the volume and the absorbance at 280 nm thereof weremeasured. The amount of salicin extracted in the upper phase (polarorganic solvent phase) was calculated from a reduction in the absorbanceat 280 nm of the lower phase and the solution volume of the lower phase.As a result, it was found that by a single extraction operation, 96.5%of the starting amount of salicin was extracted in the tetrahydrofuranphase.

Example 5b

This additional extraction operation to Example 5a was conducted exceptfor use of 10 ml of acetonitrile instead of 10 ml of tetrahydrofuran. Asa result, it was found that by a single extraction operation, 98.1% ofthe starting amount of salicin was extracted in the acetonitrile phase.

Example 6 Extraction of Salicin Example 6a

1 g of salicin was dissolved in 100 ml of an aqueous solution containing30% glucose and 10% sodium chloride to obtain a sample aqueous solution.The absorbance at 280 nm of the sample aqueous solution was measured. 10ml of acetone was added to 10 ml of the sample aqueous solution,followed by vigorous stirring using a separating funnel for 5 minutes.Thereafter, the mixture was allowed to stand for 30 minutes. As aresult, the mixture was separated into two phases. The lower phase(aqueous phase) was recovered, and the volume and the absorbance at 280nm thereof were measured. The amount of salicin extracted in the upperphase (polar organic solvent phase) was calculated from a reduction inthe absorbance at 280 nm of the lower phase and the solution volume ofthe lower phase. As a result, it was found that by a single extractionoperation, 58.0% of the starting amount of salicin was extracted in theacetone phase.

Example 6b

This additional extraction operation to Example 6a was conducted exceptfor use of 10 ml of isopropanol instead of 10 ml of acetone. As aresult, it was found that by a single extraction operation, 97.0% of thestarting amount of salicin was extracted in the isopropanol phase.

Example 7 Extraction of Caffeic Acid Example 7a

1 g of caffeic acid was dissolved in 100 ml of an aqueous solutioncontaining 30% glucose to obtain a sample aqueous solution. Theabsorbance at 280 nm of the sample aqueous solution was measured. 10 mlof tetrahydrofuran was added to 10 ml of the sample aqueous solution,followed by vigorous stirring using a separating funnel for 5 minutes.Thereafter, the mixture was allowed to stand for 30 minutes. As aresult, the mixture was separated into two phases. The lower phase(aqueous phase) was recovered, and the volume and the absorbance at 280nm thereof were measured. The amount of caffeic acid extracted in theupper phase (polar organic solvent phase) was calculated from areduction in the absorbance at 280 nm of the lower phase and thesolution volume of the lower phase. As a result, it was found that by asingle extraction operation, 88.6% of the starting amount of caffeicacid was extracted in the tetrahydrofuran phase.

Example 7b

This additional extraction operation to Example 7a was conducted exceptfor use of 10 ml of acetonitrile instead of 10 ml of tetrahydrofuran. Asa result, it was found that by a single extraction operation, 76.2% ofthe starting amount of caffeic acid was extracted in the acetonitrilephase.

Example 8 Extraction of Caffeic Acid Example 8a

1 g of caffeic acid was dissolved in 100 ml of an aqueous solutioncontaining 30% glucose and 10% sodium chloride to obtain a sampleaqueous solution. The absorbance at 280 nm of the sample aqueoussolution was measured. 10 ml of acetone was added to 10 ml of the sampleaqueous solution, followed by vigorous stirring using a separatingfunnel for 5 minutes. Thereafter, the mixture was allowed to stand for30 minutes. As a result, the mixture was separated into two phases. Thelower phase (aqueous phase) was recovered, and the volume and theabsorbance at 280 nm thereof were measured. The amount of caffeic acidextracted in the upper phase (polar organic solvent phase) wascalculated from a reduction in the absorbance at 280 nm of the lowerphase and the solution volume of the lower phase. As a result, it wasfound that by a single extraction operation, 78.2% of the startingamount of caffeic acid was extracted in the acetone phase.

Example 8b

This additional extraction operation to Example 8a was conducted exceptfor use of 10 ml of isopropanol instead of 10 ml of acetone. As aresult, it was found that by a single extraction operation, 87.2% of thestarting amount of caffeic acid was extracted in the isopropanol phase.

Example 9 Extraction of Salicyl Alcohol Example 9a

1 g of salicyl alcohol was dissolved in 100 ml of an aqueous solutioncontaining 30% glucose to obtain a sample aqueous solution. Theabsorbance at 280 nm of the sample aqueous solution was measured. 10 mlof tetrahydrofuran was added to 10 ml of the sample aqueous solution,followed by vigorous stirring using a separating funnel for 5 minutes.Thereafter, the mixture was allowed to stand for 30 minutes. As aresult, the mixture was separated into two phases. The lower phase(aqueous phase) was recovered, and the volume and the absorbance at 280nm thereof were measured. The amount of salicyl alcohol extracted in theupper phase (polar organic solvent phase) was calculated from areduction in the absorbance at 280 nm of the lower phase and thesolution volume of the lower phase. As a result, it was found that by asingle extraction operation, 98.7% of the starting material of salicylalcohol was extracted in the tetrahydrofuran phase.

Example 9b

This additional extraction operation to Example 9a was conducted exceptfor use of 10 ml of acetonitrile instead of 10 ml of tetrahydrofuran. Asa result, it was found that by a single extraction operation, 98.2% ofthe starting amount of salicyl alcohol was extracted in the acetonitrilephase.

Example 10 Extraction of Salicyl Alcohol Example 10a

0.1 g of salicyl alcohol was dissolved in 100 ml of an aqueous solutioncontaining 30% glucose and 10% sodium chloride to obtain a sampleaqueous solution. The absorbance at 280 nm of the sample aqueoussolution was measured. 10 ml of acetone was added to 10 ml of the sampleaqueous solution, followed by vigorous stirring using a separatingfunnel for 5 minutes. Thereafter, the mixture was allowed to stand for30 minutes. As a result, the mixture was separated into two phases. Thelower phase (aqueous phase) was recovered, and the volume and theabsorbance at 280 nm thereof were measured. The amount of salicylalcohol extracted in the upper phase (polar organic solvent phase) wascalculated from a reduction in the absorbance at 280 nm of the lowerphase and the solution volume of the lower phase. As a result, it wasfound that by a single extraction operation, 87.7% of the startingamount of salicyl alcohol was extracted in the acetone phase.

Example 10b

This additional extraction operation to Example 10a was conducted exceptfor use of 10 ml of isopropanol instead of 10 ml of acetone. As aresult, it was found that by a single extraction operation, 98.7% of thestarting amount of salicyl alcohol was extracted in the isopropanolphase.

Example 11 Extraction of Elladitannin and Polymers Thereof Example 11a

An aqueous sweet tea extract solution (SUNTENCHA; manufactured bySuntory Ltd.) was diluted two-fold with water to obtain a two-folddiluent. Glucose and sodium chloride were added and dissolved in thetwo-fold diluent to concentrations of 10% and 10%, respectively toobtain an aqueous solution. The absorbance at 280 nm of the aqueoussolution was measured. 20 ml of acetone was added to 20 ml of theaqueous solution, followed by vigorous stirring using a separatingfunnel for 5 minutes. Thereafter, the mixture was allowed to stand for30 minutes. As a result, the mixture was separated into two phases. Thelower phase (aqueous phase) was recovered, and the volume and theabsorbance at 280 nm thereof were measured. The amount of effectiveingredients (i.e., elladitannin and polymers thereof) extracted in theupper phase (polar organic solvent phase) was calculated from areduction in the absorbance at 280 nm of the lower phase and thesolution volume of the lower phase. As a result, it was found that by asingle extraction operation, 43.1% of the effective ingredients wereextracted in the acetone phase. Moreover, the level of coloration in theacetone phase was about ⅓ that of the aqueous phase. Thus, it was foundthat by the extraction operation, the effective ingredients wereextracted and the decolorization effect was obtained.

Example 12 Extraction of Hesperidin from Fruit Juice

10 ml of 15% sodium chloride solution was added to 10 ml of Californiaorange juice concentrate (five-fold concentrate), and stirred tohomogeneity, thereby obtaining an aqueous solution. The amount ofhesperidin in the California orange juice concentrate used was measuredby HPLC using a column ODS, where the mobile phase was a mixture ofacetonitrile and water at 20:80, the flow rate was 0.5 ml/min, and thecolumn temperature was 40° C. The absorbance of the eluate was detectedat 280 nm. A specific method of detecting the amount of hesperidin isdescribed in Japanese Laid-Open Publication No. 8-80177. 20 ml ofacetone was added to 20 ml of the resultant aqueous solution, followedby vigorous stirring using a separating funnel for 5 minutes.Thereafter, the mixture was allowed to stand for 30 minutes. As aresult, the mixture was separated into two phases. The upper phase(polar organic solvent phase) was recovered, and the volume thereof wasmeasured. The amount of hesperidin was measured by HPLC as describedabove. As a result, by a single extraction operation, 75% of hesperidinwas extracted in the acetone phase.

Example 13 Extraction of Hesperidin from Glycosylation Reaction Solution

An aqueous solution in which 5% soluble starch (manufactured by Merck)and 0.5% hesperidin were dissolved was adjusted with hydrochloric acidto pH 9.0. Alkaline-resistant CGTase (described in Japanese Laid-OpenPublication No. 7-107972) was added to the aqueous solution to aconcentration of 5 units/ml. Thereafter, an enzyme reaction was allowedto proceed at 37° C. for 16 hours. After the end of the enzyme reaction,a portion of the enzyme reaction solution was collected, and the amountsof hesperidin and hesperidin glycosides were measured by a HPLC analysismethod. In the HPLC analysis method, LiChrospher 100RP18 (Merck; 4.0×250mm) column was used, where the mobile phase was a mixture ofacetonitrile and water at 20:80, the flow rate was 0.5 ml/min, and thecolumn temperature was 40° C. The absorbance of the eluate was detectedat 280 nm. A method of analyzing hesperidin and hesperidin glycosides isdescribed in Japanese Laid-Open Publication No. 8-80177. As a result, itwas found that due to the enzyme reaction, about 80% of the hesperidinwhich was added at the start of the reaction was converted intohesperidin glycosides.

Thereafter, glucose and sodium chloride were added and dissolved in theenzyme reaction solution after the enzyme reaction to concentration of20% and 10%, respectively, to obtain a glucose and sodiumchloride-containing enzyme reaction solution. An equal volume of acetonewas added to the glucose and sodium chloride-containing enzyme reactionsolution, followed by vigorous stirring using a separating funnel for 5minutes. The mixture was allowed to stand for 30 minutes. As a result,the mixture was separated into two phases. The upper acetone phase wascollected, and the volume thereof was measured, and the amount ofhesperidin glycosides dissolved in the acetone phase was measured byHPLC as described above. As a result, about 45% of the hesperidinglycosides present in the enzyme reaction solution after the end of theenzyme reaction was extracted in the acetone phase. Further, an amountof acetone, which was half the volume of the enzyme reaction solution,was added to the lower aqueous phase, stirred, and allowed to stand, andthe upper phase was recovered. This additional extraction operation wascarried out three times. As a result, all of the hesperidin glycosidescould be recovered.

Example 14 Extraction of Hydroquinone Glycoside from GlycosylationReaction Solution

An aqueous solution, in which 35% dextrin (PINE-DEX #1 where DE is 7 to9; manufactured by Matsutani Chemical Industry Co., Ltd.) and 15%hydroquinone were dissolved, was adjusted with SN sodium hydroxideaqueous solution to pH 6.5. A glycosylation enzyme (amylase X-23described in Japanese Laid-Open Publication No. 6-277053) was added tothe aqueous solution to a concentration of 20 units/ml. Thereafter,incubation was performed at 45° C. for 40 hours to cause a hydroquinoneglycosylation reaction. After the end of the enzyme reaction, a portionof the enzyme reaction solution was collected, and the amounts ofhydroquinone and hydroquinone glycoside were measured by a HPLC analysismethod. In the HPLC analysis method, LiChrospher 100RP18 (Merck; 4.0×250mm) column was used, where the mobile phase was a mixture of water,methanol and phosphoric acid at 80:19.7:0.3 (v/v), the flow rate was 0.5ml/min, and the column temperature was 40° C. The amounts ofhydroquinone and hydroquinone glycoside in the eluate were detected byultraviolet spectroscopy. The amount of product malto-oligosaccharidewas measured by a HPLC analysis method using the enzyme reactionsolution. In the HPLC analysis method, LiChrosorb NH₂ (Merck; 4.0×250mm) column was used, where the mobile phase was a mixture of water andacetonitrile at 25:75, the flow rate was 1.0 ml/min, and the columntemperature was 40° C. Malto-oligosaccharide in the eluate was detectedby a RI detector. As a result, it was found that due to the enzymereaction, about 35% of the hydroquinone was converted into hydroquinoneglycoside, and the dextrin was degraded to glucose andmalto-oligosaccharides. Further, glucoamylase (manufactured by NagaseChemtex; brand name XL-4) was added to the enzyme reaction solutionafter the end of the enzyme reaction to a concentration of 8.8 units/ml,followed by incubation at 45° C. for 3 hours, thereby degrading theoligosaccharides in the enzyme reaction solution to glucose.

Thereafter, an equal volume of tetrahydrofuran was added to the enzymereaction solution after the end of the degradation of theoligosaccharides, followed by vigorous stirring using a separatingfunnel for 5 minutes. The mixture was allowed to stand for 30 minutes.As a result, the mixture was separated into two phases. The uppertetrahydrofuran phase was recovered, and the volume thereof wasmeasured. The amount of hydroquinone glycoside dissolved in thetetrahydrofuran phase was analyzed by HPLC as described above. As aresult, about 65% of the hydroquinone glycoside present in the enzymereaction solution after the end of the enzyme reaction was extracted inthe tetrahydrofuran phase. Further, an amount of tetrahydrofuran, whichwas half the volume of the enzyme reaction solution, was added to thelower aqueous phase, stirred, and allowed to stand, and the upper phasewas recovered. This additional extraction operation was carried outthree times. As a result, all of the hydroquinone glycoside could berecovered.

Example 15 Extraction of Hydroquinone Glycoside from GlycosylationReaction Solution

An aqueous solution, in which 35% dextrin (PINE-DEX #1 where DE is 7 to9; manufactured by Matsutani Chemical Industry Co., Ltd.) and 15%hydroquinone were dissolved, was adjusted with 5N sodium hydroxideaqueous solution to pH 6.5. A glycosylation enzyme (amylase X-23described in Japanese Laid-Open Publication No. 6-277053) was added tothe aqueous solution to a concentration of 20 units/ml. Thereafter,incubation was performed at 45° C. for 40 hours to cause a hydroquinoneglycosylation reaction. After the end of the enzyme reaction, a portionof the enzyme reaction solution was collected, and the amounts ofhydroquinone and hydroquinone glycoside were measured by a HPLC analysismethod. In the HPLC analysis method, LiChrospher 100RP18 (Merck; 4.0×250mm) column was used, where the mobile phase was a mixture of water,methanol and phosphoric acid at 80:19.7:0.3 (v/v), the flow rate was 0.5ml/min, and the column temperature was 40° C. The amounts ofhydroquinone and hydroquinone glycoside in the eluate were detected byultraviolet spectroscopy. The amount of product malto-oligosaccharidewas measured by a HPLC analysis method using the enzyme reactionsolution. In the HPLC analysis method, LiChrosorb NH₂ (Merck; 4.0×250mm) column was used, where the mobile phase was a mixture of water andacetonitrile at 25:75, the flow rate was 1.0 ml/min, and the columntemperature was 40° C. Malto-oligosaccharide in the eluate was detectedby a RI detector. As a result, it was found that due to the enzymereaction, about 35% of the hydroquinone was converted into hydroquinoneglycoside, and the dextrin was degraded to glucose andmalto-oligosaccharides. Further, glucoamylase (manufactured by NagaseChemtex; brand name XL-4) was added to the enzyme reaction solutionafter the end of the enzyme reaction to a concentration of 8.8 units/ml,followed by incubation at 45° C. for 3 hours, thereby degrading theoligosaccharides in the enzyme reaction solution to glucose.

Thereafter, the enzyme reaction solution after the degradation of theoligosaccharides was vacuum concentrated by a factor of about 1.4 usingan evaporator. Ammonium sulfate was added to the concentrate to aconcentration of 20% to obtain an ammonium sulfate-containingconcentrate. An equal volume of acetone was added to the ammoniumsulfate-containing concentrate, followed by vigorous stirring using aseparating funnel for 5 minutes. The mixture was allowed to stand for 30minutes. As a result, the mixture was separated into two phases. Theupper acetone phase was recovered, and the volume thereof was measured.The amount of hydroquinone glycoside dissolved in the acetone phase wasanalyzed by HPLC as described above. As a result, about 60% of thehydroquinone glycoside present in the enzyme reaction solution after theend of the enzyme reaction was extracted in the acetone phase. Further,an amount of acetone, which was 0.8 times the volume of the concentrate,was added to the lower aqueous phase, stirred, and allowed to stand, andthe upper phase was recovered. This additional extraction operation wascarried out three times. As a result, all of the hydroquinone glycosidecould be recovered.

Example 16 Extraction of Hydroquinone Glycoside from GlycosylationReaction Solution

An aqueous solution, in which 35% dextrin (PINE-DEX #1 where DE is 7 to9; manufactured by Matsutani Chemical Industry Co., Ltd.) and 15%hydroquinone were dissolved, was adjusted with 5N sodium hydroxideaqueous solution to pH 6.5. A glycosylation enzyme (amylase X-23described in Japanese Laid-Open Publication No. 6-277053) was added tothe aqueous solution to a concentration of 20 units/ml. Thereafter,incubation was performed at 45° C. for 40 hours to cause a hydroquinoneglycosylation reaction. After the end of the enzyme reaction, a portionof the enzyme reaction solution was collected, and the amounts ofhydroquinone and hydroquinone glycoside were measured by a HPLC analysismethod. In the HPLC analysis method, LiChrospher 100RP18 (Merck; 4.0×250mm) column was used, where the mobile phase was a mixture of water,methanol and phosphoric acid at 80:19.7:0.3 (v/v), the flow rate was 0.5ml/min, and the column temperature was 40° C. The amounts ofhydroquinone and hydroquinone glycoside in the eluate were detected byultraviolet spectroscopy. The amount of product malto-oligosaccharidewas measured by a HPLC analysis method using the enzyme reactionsolution. In the HPLC analysis method, LiChrosorb NH₂ (Merck; 4.0×250mm) column was used, where the mobile phase was a mixture of water andacetonitrile at 25:75, the flow rate was 1.0 ml/min, and the columntemperature was 40° C. Malto-oligosaccharide in the eluate was detectedby a RI detector. As a result, it was found that due to the enzymereaction, about 35% of the hydroquinone was converted into hydroquinoneglycoside, and the dextrin was degraded to glucose andmalto-oligosaccharides. Further, glucoamylase (manufactured by NagaseChemtex; brand name XL-4) was added to the enzyme reaction solutionafter the end of the enzyme reaction to a concentration of 8.8 units/ml,followed by incubation at 45° C. for 3 hours, thereby degrading theoligosaccharides in the enzyme reaction solution to glucose.

Thereafter, an equal volume of acetonitrile was added to the enzymereaction solution after the degradation of the oligosaccharides,followed by vigorous stirring using a separating funnel for 5 minutes.The mixture was allowed to stand for 1 hour. As a result, the mixturewas separated into two phases. The upper acetonitrile phase wasrecovered, and the amount thereof was measured. The amount ofhydroquinone glycoside dissolved in the acetonitrile phase was analyzedby HPLC as described above. As a result, about 30% of the hydroquinoneglycoside present in the enzyme reaction solution after the end of theenzyme reaction was extracted in the acetonitrile phase. Further, avolume of acetonitrile, which was equal to the volume of the enzymereaction solution, was added to the lower aqueous phase, stirred, andallowed to stand, and the upper phase was recovered. This additionalextraction operation was carried out five times. As a result, about 90%of the hydroquinone glycoside which was present in the enzyme reactionsolution after the end of the enzyme reaction could be recovered.

Example 17 Study of Conditions for Glucoamylase Treatment

When a glycosylation enzyme reaction is conducted using dextrin orstarch as a starting material, a large amount of glucose and dextrinhaving a lower molecular weight than that of a starting material ispresent in the enzyme reaction solution after the end of the reaction.Even if the concentration of overall saccharides is the same, the higherthe mole value, the lesser the transfer of saccharide to an organicphase. Therefore, in order to efficiently degrade dextrin remainingafter the end of the glycosylation enzyme reaction to glucoses,conditions for the degradation reaction of dextrin were studied.

An aqueous solution, in which 35% dextrin (PINE-DEX #1 where DE is 7 to9; manufactured by Matsutani Chemical Industry Co., Ltd.) and 15%hydroquinone were dissolved, was adjusted with 5N sodium hydroxideaqueous solution to pH 6.5. A glycosylation enzyme (amylase X-23described in Japanese Laid-Open Publication No. 6-277053) was added tothe aqueous solution to a concentration of 20 units/ml. Thereafter,incubation was performed at 45° C. for 40 hours to cause a hydroquinoneglycosylation reaction.

1.4 μl (1-fold amount), 2.1 μl (1.5-fold amount) or 2.8 μl (2-foldamount) of glucoamylase (manufactured by Nagase Chemtex; brand nameXL-4) was added to 1 ml of the glycosylation reaction solution after theend of the reaction. Thereafter, the mixture was incubated at 45° C. for3 or 4 hours. After the incubation, the contents of glucose and maltosein the solution were confirmed by a HPLC analysis method. In the HPLCanalysis method, LiChrosorb NH₂ (Merck; 4.0×250 mm) column was used,where the mobile phase was a mixture of water and acetonitrile at 25:75,the flow rate was 1.0 ml/mm, and the column temperature was 40° C.Glucose and maltose in the eluate were detected by a RI detector. Theamounts of hydroquinone and hydroquinone glycoside in the eluate weredetected by a HPLC analysis method. In the HPLC analysis method,LiChrospher 100RP18 (Merck; 4.0×250 mm) column was used, where themobile phase was a mixture of water, methanol and phosphoric acid at80:19.7:0.3 (v/v), the flow rate was 0.5 ml/min, and the columntemperature was 40° C. The amounts of hydroquinone and hydroquinoneglycoside in the eluate were detected by ultraviolet spectroscopy. Basedon the resultant amounts of the obtained hydroquinone and hydroquinoneglycoside (HQG), the hydroquinone glycosylation rate was calculated. Theresults are shown in Table 8 below.

TABLE 8 Glucose Maltose HQG concentration concentration glycosylation(%) (%) rate (%) Samples 3 hrs 4 hrs 3 hrs 4 hrs 3 hrs 4 hrs (XL-4) 11.612.2 5.2 4.9 36.1 36.2 1-fold (XL-4) 13.9 14.7 4.2 3.7 36.1 36.21.5-fold (XL-4) 15.2 15.8 3.0 2.6 36.1 36.2 2-fold

Even when saccharide degradation was performed using the 1-fold amountof glucoamylase for 3 or 4 hours, the enzyme reaction solution hadsubstantially the same composition as before the addition ofglucoamylase. Therefore, it was suggested that with 1-fold glucoamylase,extension of a reaction by about one or two hours is not likely todramatically reduce the amount of maltose.

Further, even if the amount of glucoamylase was increased, theglycosylation rate was not substantially changed. This indicates thatglucoamylase can degrade dextrin without degrading hydroquinoneglycoside which is a product of interest. Therefore, it was found thatfor promotion of dextrin degradation, an increase in the amount ofglucoamylase is more effective than extension of degradation reactiontime.

Next, an influence of a difference between glucoamylase treatments onTHF treatment was studied. 1 ml of (XL-4) treatment solution and 1 ml ofTHF were mixed together, followed by vigorous shaking using a separatingfunnel for 1 minute. After shaking, the mixture was allowed to stand for1 hour. As a result, the mixture was separated into two phases. Theaqueous phase was recovered. Water was added to the aqueous phase to 1ml. The amounts of glucose and maltose were measured by an HPLC analysismethod using the aqueous phase. In the HPLC analysis method, LiChrosorbNH₂ (Merck; 4.0×250 mm) column was used, where the mobile phase was amixture of water and acetonitrile at 25:75, the flow rate was 1.0ml/min, and the column temperature was 40° C. Glucose and maltose in theeluate were detected by a RI detector. The amounts of hydroquinone andhydroquinone glycoside in the eluate were measured by a HPLC analysismethod. In the HPLC analysis method, LiChrospher 100RP18 (Merck; 4.0×250mm) column was used, where the mobile phase was a mixture of water,methanol and phosphoric acid at 80: 19.7:0.3 (v/v), the flow rate was0.5 ml/mm, and the column temperature was 40° C. The amounts ofhydroquinone and hydroquinone glycoside in the eluate were measured byultraviolet spectroscopy. Based on the obtained results and theabove-described glucose concentration, maltose concentration and HQGconcentration before extraction with THF, the extraction rate to the THFphase was calculated. The results are shown in Table 9 below.

TABLE 9 Extraction rate to THF (%) Samples Glucose Maltose HQG (XL-4)43.3 38.6 71.2 1-fold (XL-4) 36.3 33.0 70.0 1.5-fold (XL-4) 38.0 36.473.7 2-fold

As a result, it was confirmed that as the glucose concentration wasincreased by the glucoamylase treatment, the extraction of glucose tothe THF phase tended to be suppressed.

A tendency was also confirmed, in which the larger the amount ofglucoamylase added to the sample, the smaller the volume of the aqueousphase recovered after shaking with THF and separation (the smaller theamount of water transferred to the THF phase).

Example 18 Influence of Saccharide Concentration on Extraction to aTetrahydrofuran Phase

An influence of glucose concentration in an aqueous solution onextraction to a tetrahydrofuran (THF) phase was studied.

Initially, an aqueous solution containing 42% glucose and 9%hydroquinone glycoside was prepared. This aqueous solution was seriallydiluted with water to prepare aqueous solutions having glucoseconcentrations from 40% to 20%. Needless to say, by the dilution of theaqueous solution, not only glucose but also hydroquinone glycoside wasdiluted. 5 ml of THF was added to 5 ml of the thus-prepared aqueoussolution, followed by stirring at 30° C. for 5 minutes using a mixer.After the stirring, the mixture was centrifuged at 3000 rpm for 5minutes. The upper phase was recovered. As the glucose concentrationdecreased, the amount of the THF phase was lessened. When the glucoseconcentration was 20%, phase separation did not occur by a singleextraction operation. 5 ml of THF was added to the lower phase again andthen the upper phase was similarly recovered. The two upper phasesobtained for the aqueous solutions was added together. The contents ofglucose and maltose were measured by a HPLC analysis method. In the HPLCanalysis method, LiChrosorb NH₂ (Merck; 4.0×250 mm) column was used,where the mobile phase was a mixture of water and acetonitrile at 25:75,the flow rate was 1.0 ml/min, and the column temperature was 40° C.Glucose and maltose in the eluate were detected by a RI detector. Theamounts of hydroquinone and hydroquinone glycoside in the eluate weredetected by a HPLC analysis method. In the HPLC analysis method,LiChrospher 100RP18 (Merck; 4.0×250 mm) column was used, where themobile phase was a mixture of water, methanol and phosphoric acid at80:19.7:0.3 (v/v), the flow rate was 0.5 ml/min, and the columntemperature was 40° C. The amounts of hydroquinone and hydroquinoneglycoside in the eluate were measured by ultraviolet spectroscopy.

The results are shown in FIG. 1. As can be seen from FIG. 1, thetransfer rate of hydroquinone glycoside was not much dependent on theglucose concentration. However, the transfer rate of glucose decreasedwith an increase in the glucose concentration. Accordingly, it was foundthat if the saccharide concentration of an aqueous solution for use inextraction is increased, the transfer of a saccharide to a polar organicsolvent phase is less than the transfer of a hydrophobicgroup-containing water-soluble organic compound, whereby a high-purityhydrophobic group-containing water-soluble organic compound can beobtained.

Example 19 Influence of Concentration of Glycosylation Reaction Solutionon Extraction of Hydroquinone Glycoside

As shown in the above-described Example 18, it was found that if thesaccharide concentration of an aqueous solution is increased, thetransfer of the saccharide to an organic phase is suppressed, so that ahydrophobic group-containing water-soluble organic compound isefficiently transferred to the organic phase. Therefore, it wasconfirmed whether or not by concentrating an enzyme reaction solutioncontaining a hydrophobic group-containing water-soluble organiccompound, the transfer of a saccharide to an organic phase wassuppressed, so that the extraction efficiency of the hydrophobicgroup-containing water-soluble organic compound was increased.

An aqueous solution, in which 35% dextrin (PINE-DEX #1 where DE is 7 to9; manufactured by Matsutani Chemical Industry Co., Ltd.) and 15%hydroquinone were dissolved, was adjusted with 5N sodium hydroxideaqueous solution to pH 6.5. A glycosylation enzyme (amylase X-23described in Japanese Laid-Open Publication No. 6-277053) was added tothe aqueous solution to a concentration of 20 units/ml. Therefore,incubation was performed at 45° C. for 40 hours to cause a hydroquinoneglycosylation reaction. 2.1 μl (1.5-fold amount) of glucoamylase(manufactured by Nagase Chemtex; brand name XL-4) was added per ml ofthe glycosylation reaction solution after the end of the reaction.Thereafter, glucoamylase treatment was performed at 45° C. for 4 hours,thereby obtaining a reaction solution.

5 ml of this reaction solution was condensed by an evaporator to beconcentrated to 80% (4.0 ml) or 70% (3.5 ml) by volume. An equal volumeof THF was added to an unconcentrated reaction solution, the 80%concentrate or the 70% concentrate, followed by vigorous shaking using amixer for 1 minute. Extraction was conducted under a condition of 30° C.The mixture was allowed to stand for 1 hour. The mixture was separatedinto two phases. The upper THF phase was recovered. The resultant THFphase was analyzed by HPLC as described above for the contents ofglucose, maltose and hydroquinone glycoside. A step of adding an amountof THF, which was 0.5 times the amount of the aqueous phase, to thelower aqueous phase again, shaking, extraction, and recovering the upperTHF phase, was further repeated twice. The obtained THF phases werecombined together. Note that although an intermediate layer wasgenerated in the concentrate during the phase separation, theintermediate layer was included in the aqueous phase. The results areshown in the following Tables 10 to 12.

TABLE 10 Unconcentrated amylase treatment reaction solution (Brixconcentration of two-fold diluent (Bx) = 24.5) Volume of Correctedconcentration solution (equivalent in 5 ml) % Extraction rate (%)Samples (ml) Glucose Maltose HQG Glucose Maltose HQG Unconcentrated 518.8 1.7 10.8 amylase treatment reaction solution THF phase total 9.57.4 0.1 9.9 39.2 3.2 90.9

TABLE 11 80% concentrate (two-fold diluent Bx = 29.5) (glucose 22.7%,maltose: 1.3%, HQG: 13.5%) Volume of Corrected concentration solution(equivalent in 5 ml) % Extraction rate (%) Samples (ml) Glucose MaltoseHQG Glucose Maltose HQG GA treatment 4 18.2 1.1 10.8 solution 80%concentrate THF phase total 9.2 4.7 0 10.9 26.1 0 101.1

TABLE 12 70% concentrate (two-fold diluent Bx = 34.0) (glucose 25.8%,maltose: 1.9%, HQG: 15.2%) Volume of Corrected concentration solution(equivalent in 5 ml) % Extraction rate (%) Samples (ml) Glucose MaltoseHQG Glucose Maltose HQG GA treatment 3.5 18 1.3 10.6 solution 70%concentrate THF phase total 7.7 3.9 0 10.1 21.7 0 95.2

As a result, as expected, by performing THF extraction afterconcentrating the post-glucoamylase treatment reaction solution, theamount of glucose extracted in the THF phase could be suppressed toabout 50% to 60% of that extracted from the unconcentrated solution.

Example 20 Influence of a Hydrophobic Group-containing Water-solubleOrganic Compound on Phase Separation

As described in Example 17, when a glycosylation reaction solution wassubjected to extraction using THF, an aqueous phase was completelyseparated from the organic phase even if the glucose concentration wasno more than 20%, and the THF phase was larger than that of the aqueousphase. On the other hand, in the above-described Example 18, when asolution containing 42% glucose and 9% hydroquinone glycoside(containing substantially no hydroquinone) was diluted with water to aglucose concentration of 20%, phase separation was not obtained in spiteof addition of THF. Therefore, an influence of a hydrophobicgroup-containing water-soluble organic compound on phase separation wasinvestigated.

Hydroquinone was added to a 20% glucose-containing diluent as preparedin the above-described Example 18, to a concentration of 10%, therebyobtaining an aqueous solution. An equal volume of THF was added to theaqueous solution, followed by stirring using a mixer for 5 minutes.Thereafter, the mixture was allowed to stand for 1 hour. As a result, aTHF phase was completely separated from the aqueous phase. Moreover,water got mixed in the THF phase, so that the THF phase was larger thanthe aqueous phase. The THF phase was recovered. The content of ahydroquinone glycoside in THF was measured by HPLC as described above.As a result, it was found that a larger amount of hydroquinone glycosidewas extracted than when hydroquinone was not added. The reason isbelieved to be that by adding a substance well soluble in a THF phase,such as hydroquinone, to an aqueous phase, THF is not easily transferredto the aqueous phase, so that separation was more efficient and a highconcentration of hydroquinone was dissolved in THF so that thehydroquinone promoted the transfer of the glycoside.

Example 21 Influence of Temperature on Extraction to a TetrahydrofuranPhase

The influence of temperature on extraction to a tetrahydrofuran phasewas investigated.

Initially, an aqueous solution, in which 35% dextrin (PINE-DEX #1 whereDE is 7 to 9; manufactured by Matsutani Chemical Industry Co., Ltd.) and15% hydroquinone were dissolved, was adjusted with 5N sodium hydroxideaqueous solution to pH 6.5. A glycosylation enzyme (amylase X-23described in Japanese Laid-Open Publication No. 6-277053) was added tothe aqueous solution to a concentration of 20 units/ml. Thereafter,incubation was performed at 45° C. for 40 hours to cause a hydroquinoneglycosylation reaction. After the end of the reaction, 2.1 μl (1.5-foldamount) of glucoamylase (manufactured by Nagase Chemtex; brand nameXL-4) was added to each 1 ml of the glycosylation reaction solution.Thereafter, the mixture was incubated at 45° C. for 4 hours to obtain apost-glucoamylase treatment reaction solution.

5 ml of THF was added to 5 ml of the post-glucoamylase treatmentreaction solution, followed by stirring using a mixer at 3, 10, 22, or45° C. for 5 minutes. Thereafter, the mixture was centrifuged at 3000rpm for 5 minutes. As a result, the mixture was separated into twophases. The aqueous phase was recovered. Water was added to the aqueousphase to a volume of 5 ml to obtain an aqueous solution. After theadjustment, the hydroquinone, hydroquinone glycoside and glucoseconcentrations of the aqueous solution were measured by HPLC asdescribed above.

The results are shown in Table 13.

TABLE 13 the hydroquinone, hydroquinone glycoside and glucoseconcentrations of the aqueous phase Start 3° C. 10° C. 20° C. 22° C. 30°C. 45° C. HQ 9.5 0.9 0.7 1.0 1.0 1.0 1.2 HQG 11.5 2.8 2.7 3.4 3.5 3.54.8 Glucose 19.4 11.1 11.5 13.3 13.4 13.4 16.6 unit: %

Based on the results in Table 13, the transfer rate of hydroquinoneglycoside and glucose were calculated. FIG. 2 shows a graph of thetransfer rate.

As can be seen from Table 13 and FIG. 2, when the extraction temperaturewas low, a large amount of saccharide was transferred to the THF phase.As the temperature was increased, the transfer rates of hydroquinone,hydroquinone glycoside and glucose to the THF phase were decreased. Thereason is believed to be that the solubility of HQG and glucose to theaqueous phase increased with an increase in temperature, so that thetransfer to the THF phase was decreased. Further, the higher thetemperature, the larger the difference between the transfer rates ofhydroquinone glycoside and glucose. Therefore, it was found that thepurity of hydroquinone glycoside can be more increased by performingextraction at as high a temperature as possible.

INDUSTRIAL APPLICABILITY

According to the present invention, a method of extracting a hydrophobicgroup-containing water-soluble organic compound is provided. Theextracting method and the purifying method of the present invention canbe used as a technique for extracting and purifying an effectiveingredient from various animals and plants, or as a technique ofextracting and purifying an effective ingredient from various enzymereaction solutions. According to the method of the present invention, ahydrophobic group-containing water-soluble organic compound can beeasily and inexpensively separated and purified.

1. A method for extracting a hydrophobic group-containing water-solubleorganic compound, comprising the step of: bringing an aqueous solutioncontaining the hydrophobic group-containing water-soluble organiccompound and a saccharide into contact with a polar organic solvent toobtain an aqueous phase and an organic phase, whereby the hydrophobicgroup-containing water-soluble organic compound is transferred to theorganic phase; wherein the hydrophobic group-containing water-solubleorganic compound is selected from the group consisting of hydroquinoneglycoside, catechin, hesperidin, hesperidin glycosides, caffeic acid,salicyl alcohol, and elladitannin; and wherein the polar organic solventis tetrahydrofuran or acetonitrile.
 2. A method according to claim 1,wherein the saccharide concentration of the aqueous solution is at least12 g per 100 ml of the aqueous solution.
 3. A method according to claim1, wherein the amount of the polar organic solvent is 0.2 to 2 times thevolume of the aqueous solution.
 4. A method according to claim 1,wherein the hydrophobic group-containing water-soluble organic compoundis derived from an enzyme reaction solution.
 5. A method according toclaim 4, wherein the enzyme reaction solution is a glycosylationreaction solution.
 6. A method according to claim 5, wherein theglycosylation reaction solution is a hesperidin or hydroquinoneglycosylation reaction solution.
 7. A method according to claim 1,wherein the hydrophobic group-containing water-soluble organic compoundis derived from an organism selected from animals or plants.
 8. A methodaccording to claim 1, wherein the hydrophobic group-containingwater-soluble organic compound is derived from fruit juice.
 9. A methodaccording to claim 1, wherein the aqueous solution is prepared byconcentrating an enzyme reaction solution containing the hydrophobicgroup-containing water-soluble organic compound and the saccharide. 10.A method according to claim 9, wherein the enzyme reaction solution is aglycosylation reaction solution.
 11. A method according to claim 10,wherein the glycosylation reaction solution is a hesperidin orhydroquinone glycosylation reaction solution.
 12. A method according toclaim 1, wherein the aqueous solution is prepared by concentrating ordiluting an extract of an organism, wherein the extract contains thehydrophobic group-containing water-soluble organic compound and thesaccharide, and the organism is an animal or a plant.
 13. A methodaccording to claim 1, wherein the aqueous solution is prepared byconcentrating fruit juice.
 14. A method for purifying a phenolderivative glycoside, comprising the steps of: bringing a first aqueoussolution containing a phenol derivative, a phenol derivative glycosideand a saccharide into contact with a polar organic solvent to obtain afirst aqueous phase and an organic phase containing a small amount ofwater, whereby the phenol derivative and the phenol derivative glycosideare transferred to the organic phase; recovering the organic phasecontaining the small amount of water; removing the polar organic solventfrom the organic phase containing the small amount of water to obtain asecond aqueous solution containing the phenol derivative and the phenolderivative glycoside; bringing the second aqueous solution into contactwith ethyl acetate to obtain a second aqueous phase and an ethyl acetatephase, whereby the phenol derivative is transferred to the ethyl acetatephase; recovering the second aqueous phase; and concentrating andcooling the second aqueous phase to precipitate the phenol derivativeglycoside; wherein the phenol derivative and the phenol derivativeglycoside are derived from a phenol derivative glycosylation reactionsolution; wherein the glycosylation reaction solution is a hesperidin orhydroquinone glycosylation reaction solution; and wherein the polarorganic solvent is tetrahydrofuran or acetonitrile.
 15. A methodaccording to claim 14, wherein the amount of the polar organic solventis 0.2 to 2 times the volume of the first aqueous solution.
 16. A methodaccording to claim 14, wherein the glycosylation reaction solution is ahydroquinone glycosylation reaction solution.
 17. A method forextracting a hydrophobic group-containing water-soluble organiccompound, comprising the steps of: bringing an aqueous solutioncontaining the hydrophobic group-containing water-soluble organiccompound and a saccharide into contact with a polar organic solvent toobtain an aqueous phase and an organic phase, wherein the hydrophobicgroup-containing water-soluble organic compound is transferred to theorganic phase; and separating and recovering the organic phase from theaqueous phase, wherein the hydrophobic group-containing water-solubleorganic compound is selected from the group consisting of hydroquinoneglycoside, catechin, hesperidin, hesperidin glycosides, caffeic acid,salicyl alcohol, and elladitannin; and wherein the polar organic solventis tetrahydrofuran or acetonitrile.
 18. A method according to claim 17,further comprising removing at least a portion of the polar organicsolvent from the organic phase.
 19. A method according to claim 17,wherein the saccharide concentration of the aqueous solution is at least12 g per 100 ml of the aqueous solution.
 20. A method according to claim17, wherein the amount of the polar organic solvent is 0.2 to 2 timesthe volume of the aqueous solution.
 21. A method according to claim 17,wherein the hydrophobic group-containing water-soluble organic compoundis derived from an enzyme reaction solution.
 22. A method according toclaim 21, wherein the enzyme reaction solution is a glycosylationreaction solution.
 23. A method according to claim 22, wherein theglycosylation reaction solution is a hesperidin or hydroquinoneglycosylation reaction solution.
 24. A method according to claim 17,wherein the hydrophobic group-containing water-soluble organic compoundis derived from an organism selected from animals or plants.
 25. Amethod according to claim 17, wherein the hydrophobic group-containingwater-soluble organic compound is derived from fruit juice.
 26. A methodaccording to claim 17, wherein the aqueous solution is prepared byconcentrating an enzyme reaction solution containing the hydrophobicgroup-containing water-soluble organic compound and the saccharide. 27.A method according to claim 26, wherein the enzyme reaction solution isa glycosylation reaction solution.
 28. A method according to claim 27,wherein the glycosylation reaction solution is a hesperidin orhydroquinone glycosylation reaction solution.
 29. A method according toclaim 17, wherein the aqueous solution is prepared by concentrating ordiluting an extract of an organism, wherein the extract contains thehydrophobic group-containing water-soluble organic compound and thesaccharide, and the organism is an animal or a plant.
 30. A methodaccording to claim 17, wherein the aqueous solution is prepared byconcentrating fruit juice.